Presence Of Intron Research Articles

Identification of essential intron sequences that enhance gene expression independently of splicing in the yeast Saccharomyces cerevisiae

Gene expression in eukaryotes is enhanced by the presence of introns in a process known as intron-mediated enhancement (IME), but its mechanism remains unclear. In Saccharomyces cerevisiae, sequences at the 5′-splice sites (SS) and branch point sites (BPS) are highly conserved compared with other higher eukaryotes. Here, the minimum intron sequence essential for IME was investigated using various short introns and a yeast codon-optimized luciferase gene as an IME model. Mutations at the 5′-SS conserved sequence and branch point in the QCR10 intron caused splicing deficiency with either a complete loss or a marked decrease in IME. By contrast, however, the 3′-AG to tG mutant was spliced and retained IME function. Moreover, heterologous introns, which did not show IME in S. cerevisiae, gained splicing competency and IME ability by substitutions to the S. cerevisiae-type 5′-SS and BPS sequences. Intriguingly, several deletion mutants between the 5′-SS and BPS in introns exhibited high levels of IME despite a loss in splicing competency. In most cases, further deletions or substitutions did not recover splicing competency and were found to decrease IME. However, a 16-nt variant consisting of the conserved 5′-SS and BPS sequences and 3′-CAG showed an IME level comparable with that of the wild-type intron. These results indicate that IME can be independent of splicing in S. cerevisiae while intron sequences at the 5′-SS and BPS play an essential role in IME.

Introns control stochastic allele expression bias

Monoallelic expression (MAE) or extreme allele bias can account for incomplete penetrance, missing heritability and non-Mendelian diseases. In cancer, MAE is associated with shorter patient survival times and higher tumor grade. Prior studies showed that stochastic MAE is caused by stochastic epigenetic silencing, in a gene and tissue-specific manner. Here, we used C. elegans to study stochastic MAE in vivo. We found allele bias/MAE to be widespread within C. elegans tissues, presenting as a continuum from fully biallelic to MAE. We discovered that the presence of introns within alleles robustly decreases MAE. We determined that introns control MAE at distinct loci, in distinct cell types, with distinct promoters, and within distinct coding sequences, using a 5’-intron position-dependent mechanism. Bioinformatic analysis showed human intronless genes are significantly enriched for MAE. Our experimental evidence demonstrates a role for introns in regulating MAE, possibly explaining why some mutations within introns result in disease.

Expression of a Cutinase of Moniliophthora roreri with Polyester and PET-Plastic Residues Degradation Activity.

ABSTRACTCutinases are enzymes produced by phytopathogenic fungi like Moniliophthora roreri. The three genome-located cutinase genes of M. roreri were amplified from cDNA of fungi growing in different induction culture media for cutinase production. The mrcut1 gene was expressed in the presence of a cacao cuticle, while the mrcut2 and mrcut3 genes were expressed when an apple cuticle was used as the inducer. The sequences of all genes were obtained and analyzed by bioinformatics tools to determine the presence of signal peptides, introns, glycosylation, and regulatory sequences. Also, the theoretical molecular weight and pI were obtained and experimentally confirmed. Finally, cutinase 1 from M. roreri (MRCUT1) was selected for heterologous expression in Escherichia coli. Successful overexpression of MRCUT1 was observed with the highest enzyme activity of 34,036 U/mg under the assay conditions at 40°C and pH 8. Furthermore, the degradation of different synthetic polyesters was evaluated; after 21 days, 59% of polyethylene succinate (PES), 43% of polycaprolactone (PCL), and 31% of polyethylene terephthalate (PET) from plastic residues were degraded.IMPORTANCE Plastic pollution is exponentially increasing; even the G20 has recognized an urgent need to implement actions to reduce it. In recent years, searching for enzymes that can degrade plastics, especially those based on polyesters such as PET, has been increasing as they can be a green alternative to the actual plastic degradation process. A promising option in recent years refers to biological tools such as enzymes involved in stages of partial and even total degradation of some plastics. In this context, the MRCUT1 enzyme can degrade polyesters contained in plastic residues in a short time. Besides, there is limited knowledge about the biochemical properties of cutinases from M. roreri. Commonly, fungal enzymes are expressed as inclusion bodies in E. coli with reduced activity. Interestingly, the successful expression of one cutinase of M. roreri in E. coli with enhanced activity is described.

Cues from mRNA splicing prevent default Argonaute silencing in C.elegans.

In animals, Argonaute small-RNA pathways scan germline transcripts to silence self-replicating genetic elements. However, little is known about how endogenous gene expression is recognized and licensed. Here, we show that the presence of introns and, by inference, the process of mRNA splicing prevents default Argonaute-mediated silencing in the C.elegans germline. The silencing of intronless genes is initiated independently of the piRNA pathway but nevertheless engages multiple components of the downstream amplification and maintenance mechanisms that mediate transgenerational silencing, including both nuclear and cytoplasmic members of the worm-specific Argonaute gene family (WAGOs). Small RNAs amplified from intronless mRNAs can trans-silence cognate intron-containing genes. Interestingly, a second, small RNA-independent cis-acting mode of silencing also acts on intronless mRNAs. Our findings suggest that cues put in place during mRNA splicing license germline gene expression and provide evidence for a splicing-dependent and dsRNA- and piRNA-independent mechanism that can program Argonaute silencing.

No Expression Divergence despite Transcriptional Interference between Nested Protein-Coding Genes in Mammals.

Nested protein-coding genes accumulated throughout metazoan evolution, with early analyses of human and Drosophila microarray data indicating that this phenomenon was simply due to the presence of large introns. However, a recent study employing RNA-seq data uncovered evidence of transcriptional interference driving rapid expression divergence between Drosophila nested genes, illustrating that accurate expression estimation of overlapping genes can enhance detection of their relationships. Hence, here I apply an analogous approach to strand-specific RNA-seq data from human and mouse to revisit the role of transcriptional interference in the evolution of mammalian nested genes. A genomic survey reveals that whereas mammalian nested genes indeed accrued over evolutionary time, they are retained at lower frequencies than in Drosophila. Though several properties of mammalian nested genes align with observations in Drosophila and with expectations under transcriptional interference, contrary to both, their expression divergence is not statistically different from that between unnested genes, and also does not increase after nesting. Together, these results support the hypothesis that lower selection efficiencies limit rates of gene expression evolution in mammals, leading to their reliance on immediate eradication of deleterious nested genes to avoid transcriptional interference.

Cooperative roles of introns 1 and 2 of tobacco resistance gene N in enhanced N transcript expression and antiviral defense responses

The tobacco virus resistance gene N contains four introns. Transient expression of transcripts from an N transgene containing these introns and driven by the native promoter in the presence of the elicitor of tobacco mosaic virus resulted in its increased expression. The requirement of the native promoter, the elicitor, or the individual introns for enhanced expression of N has not been fully studied. Here, we determined that 35S promoter-driven N transcript expression could be enhanced in the presence of the four introns regardless of the co-expression of the virus elicitor in tobacco. Function analyses using a series of N transgenes with different combination of introns revealed that the presence of intron 1 more so than intron 2 allowed higher accumulation of premature and mature N transcripts; however, both introns were important for not only enhanced gene expression but also for induction of cell death in tobacco and induced local resistance to spread of virus in Nicotiana benthamiana. Our findings indicate that introns 1 and 2 cooperatively contribute to N expression and virus resistance.

Evolutionary Perspective and Expression Analysis of Intronless Genes Highlight the Conservation of Their Regulatory Role.

The structure of eukaryotic genes is generally a combination of exons interrupted by intragenic non-coding DNA regions (introns) removed by RNA splicing to generate the mature mRNA. A fraction of genes, however, comprise a single coding exon with introns in their untranslated regions or are intronless genes (IGs), lacking introns entirely. The latter code for essential proteins involved in development, growth, and cell proliferation and their expression has been proposed to be highly specialized for neuro-specific functions and linked to cancer, neuropathies, and developmental disorders. The abundant presence of introns in eukaryotic genomes is pivotal for the precise control of gene expression. Notwithstanding, IGs exempting splicing events entail a higher transcriptional fidelity, making them even more valuable for regulatory roles. This work aimed to infer the functional role and evolutionary history of IGs centered on the mouse genome. IGs consist of a subgroup of genes with one exon including coding genes, non-coding genes, and pseudogenes, which conform approximately 6% of a total of 21,527 genes. To understand their prevalence, biological relevance, and evolution, we identified and studied 1,116 IG functional proteins validating their differential expression in transcriptomic data of embryonic mouse telencephalon. Our results showed that overall expression levels of IGs are lower than those of MEGs. However, strongly up-regulated IGs include transcription factors (TFs) such as the class 3 of POU (HMG Box), Neurog1, Olig1, and BHLHe22, BHLHe23, among other essential genes including the β-cluster of protocadherins. Most striking was the finding that IG-encoded BHLH TFs fit the criteria to be classified as microproteins. Finally, predicted protein orthologs in other six genomes confirmed high conservation of IGs associated with regulating neural processes and with chromatin organization and epigenetic regulation in Vertebrata. Moreover, this study highlights that IGs are essential modulators of regulatory processes, such as the Wnt signaling pathway and biological processes as pivotal as sensory organ developing at a transcriptional and post-translational level. Overall, our results suggest that IG proteins have specialized, prevalent, and unique biological roles and that functional divergence between IGs and MEGs is likely to be the result of specific evolutionary constraints.

Analysis of Fungal Genomes Reveals Commonalities of Intron Gain or Loss and Functions in Intron-Poor Species.

Previous evolutionary reconstructions have concluded that early eukaryotic ancestors including both the last common ancestor of eukaryotes and of all fungi had intron-rich genomes. By contrast, some extant eukaryotes have few introns, underscoring the complex histories of intron–exon structures, and raising the question as to why these few introns are retained. Here, we have used recently available fungal genomes to address a variety of questions related to intron evolution. Evolutionary reconstruction of intron presence and absence using 263 diverse fungal species supports the idea that massive intron reduction through intron loss has occurred in multiple clades. The intron densities estimated in various fungal ancestors differ from zero to 7.6 introns per 1 kb of protein-coding sequence. Massive intron loss has occurred not only in microsporidian parasites and saccharomycetous yeasts, but also in diverse smuts and allies. To investigate the roles of the remaining introns in highly-reduced species, we have searched for their special characteristics in eight intron-poor fungi. Notably, the introns of ribosome-associated genes RPL7 and NOG2 have conserved positions; both intron-containing genes encoding snoRNAs. Furthermore, both the proteins and snoRNAs are involved in ribosome biogenesis, suggesting that the expression of the protein-coding genes and noncoding snoRNAs may be functionally coordinated. Indeed, these introns are also conserved in three-quarters of fungi species. Our study shows that fungal introns have a complex evolutionary history and underappreciated roles in gene expression.

The Mitogenomes of Ophiostoma minus and Ophiostoma piliferum and Comparisons With Other Members of the Ophiostomatales.

Fungi assigned to the Ophiostomatales are of economic concern as many are blue-stain fungi and some are plant pathogens. The mitogenomes of two blue-stain fungi, Ophiostoma minus and Ophiostoma piliferum, were sequenced and compared with currently available mitogenomes for other members of the Ophiostomatales. Species representing various genera within the Ophiostomatales have been examined for gene content, gene order, phylogenetic relationships, and the distribution of mobile elements. Gene synteny is conserved among the Ophiostomatales but some members were missing the atp9 gene. A genome wide intron landscape has been prepared to demonstrate the distribution of the mobile genetic elements (group I and II introns and homing endonucleases) and to provide insight into the evolutionary dynamics of introns among members of this group of fungi. Examples of complex introns or nested introns composed of two or three intron modules have been observed in some species. The size variation among the mitogenomes (from 23.7 kb to about 150 kb) is mostly due to the presence and absence of introns. Members of the genus Sporothrix sensu stricto appear to have the smallest mitogenomes due to loss of introns. The taxonomy of the Ophiostomatales has recently undergone considerable revisions; however, some lineages remain unresolved. The data showed that genera such as Raffaelea appear to be polyphyletic and the separation of Sporothrix sensu stricto from Ophiostoma is justified.

Regulation of pri-MIRNA processing: mechanistic insights into the miRNA homeostasis in plant.

miRNAs in plant plays crucial role in controlling proper growth, development and fitness by modulating the expression of their target genes. Therefore to modulate the expression of any stress/development related gene specifically, it is better to modulate expression of the miRNA that can target that gene. To modulate the expression level of miRNA, it is prerequisite to uncover the underlying molecular mechanism of its biogenesis. The biogenesis pathway consists of two major steps, transcription of MIR gene to pri-MIRNA and processing of pri-MIRNA into mature miRNA via sequential cleavage steps. Both of these pathways are tightly controlled by several different factors involving structural and functional molecules. This review is mainly focused on different aspects of pri-MIRNA processing mechanism to emphasize on the fact that to modulate the level of a miRNA in the cell only over-expression or knock-down of that MIR gene is not always sufficient rather it is also crucial to take processing regulation into consideration. The data collected from the recent and relevant literatures depicts that processing regulation is controlled by several aspects like structure and size of the pri-MIRNA, presence of introns in MIR gene and their location, interaction of processing factors with the core components of processing machinery etc. These detailed information can be utilized to figure out the particular point which can be utilized to modulate the expression of the miRNA which would ultimately be beneficial for the scientist and researcher working in this field to generate protocol for engineering plant with improved yield and stress tolerance.

Overexpression of AtAHL20 causes delayed flowering in Arabidopsis via repression of FT expression

BackgroundThe 29-member Arabidopsis AHL gene family is classified into three main classes based on nucleotide and protein sequence evolutionary differences. These differences include the presence or absence of introns, type and/or number of conserved AT-hook and PPC domains. AHL gene family members are divided into two phylogenetic clades, Clade-A and Clade-B. A majority of the 29 members remain functionally uncharacterized. Furthermore, the biological significance of the DNA and peptide sequence diversity, observed in the conserved motifs and domains found in the different AHL types, is a subject area that remains largely unexplored.ResultsTransgenic plants overexpressing AtAHL20 flowered later than the wild type under both short and long days. Transcript accumulation analyses showed that 35S:AtAHL20 plants contained reduced FT, TSF, AGL8 and SPL3 mRNA levels. Similarly, overexpression of AtAHL20’s orthologue in Camelina sativa, Arabidopsis’ closely related Brassicaceae family member species, conferred a late-flowering phenotype via suppression of CsFT expression. However, overexpression of an aberrant AtAHL20 gene harboring a missense mutation in the AT-hook domain’s highly conserved R-G-R core motif abolished the late-flowering phenotype. Data from targeted yeast-two-hybrid assays showed that AtAHL20 interacted with itself and several other Clade-A Type-I AHLs which have been previously implicated in flowering-time regulation: AtAHL19, AtAHL22 and AtAHL29.ConclusionWe showed via gain-of-function analysis that AtAHL20 is a negative regulator of FT expression, as well as other downstream flowering time regulating genes. A similar outcome in Camelina sativa transgenic plants overexpressing CsAHL20 suggest that this is a conserved function. Our results demonstrate that AtAHL20 acts as a photoperiod-independent negative regulator of transition to flowering.

High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns

The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant genome. The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs, including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana, we investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLSs), and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one. However, the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence, which dramatically improved the editing efficiency of the constructs. None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes. The intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.

Investigating the Role of Introns in Transcription‐Associated Mutagenesis in Budding Yeast

A key feature required for cell survival is genomic stability, which is the ability of a cell to prevent mutations in its genome and repair them if they do occur. Genomic mutations can be generated in several ways, including transcription of a gene via transcription‐associated mutagenesis (TAM). Introns are known to be able to enhance transcription in vivo, but the influence of introns on TAM is not well‐understood. Here, our work investigates whether introns modulate the rate of TAM in a URA3 reporter gene using budding yeast as a model organism. Our preliminary results suggest that the presence of introns in the natively‐intronless URA3 gene is associated with elevated mutation rates of that gene. Interestingly, there appears to be an intron‐length‐dependent effect on this trend (i.e. the yeast strain with a long intron in the URA3 reporter demonstrated elevated URA3 gene mutation rates compared to the other two strains with either a short intron or no intron). Our findings suggest that the length and presence of introns in a gene are associated with increased mutagenesis of that gene. Furthermore, the long‐intron‐containing strains in our study grew slower in the absence of uracil than the short‐intron strains and no‐intron controls, possibly indicating an effect of intron length on splicing of the URA3 primary transcript. We are currently investigating if elevated transcription rate of a URA3 gene containing an intron causes an increase in TAM rate. In addition, URA3 reporter gene expression levels are being quantitated in each strain. We expect that URA3 mRNA transcript levels will be elevated in the intron‐containing URA3 yeast strains relative to those of the no‐intron controls. In future work for this study, we will determine the effect of the presence of an intron located near the promoter of an induced URA3 gene on the rate of TAM. Our future results will contribute to better understanding the impact of introns on the mutagenesis of highly‐expressed genes, which may play a role in mechanisms underlying mutagenesis in the human genome.Support or Funding InformationDr. Jane Kim’s research is supported by an NIH SCORE Grant (SC3GM127198).

The Tc1-like elements with the spliceosomal introns in mollusk genomes.

Transposable elements (TEs) are DNA sequences capable of transpositions within the genome and thus exerting a considerable influence on the genome functioning and structure and serving as a source of new genes. TE biodiversity studies in previously unexplored species are important for the fundamental understanding of the TE influence on eukaryotic genomes. TEs are classified into retrotransposons and DNA transposons. IS630/Tc1/mariner (ITm) superfamily of DNA transposons is one of the most diverse groups broadly represented among the eukaryotes. The study of 19 mollusk genomes revealed a new group of ITm superfamily elements, which we henceforth refer to as TLEWI. These TEs are characterized by the low copy number, the lack of terminal inverted repeats, the catalytic domain with DD36E signature and the presence of spliceosomal introns in transposase coding sequence. Their prevalence among the mollusks is limited to the class Bivalvia. Since TLEWI possess the features of domesticated TE and structures similar to the eukaryotic genes which are not typical for the DNA transposons, we consider the hypothesis of co-optation of TLEWI gene by the bivalves. The results of our study will fill the gap of knowledge about the prevalence, activity, and evolution of the ITm DNA transposons in multicellular genomes and will facilitate our understanding of the mechanisms of TE domestication by the host genome.

Enhancing the yield of human erythropoietin in Aspergillus niger by introns and CRISPR-Cas9

Aspergillus niger has been employed to produce heterologous proteins due to its high capacity for expression and secretion; nevertheless, expression levels of human proteins have been modest. We were interested in investigating whether A. niger can express and secret human erythropoietin (HuEPO) at high yields. Our strategy was to combine the presence of introns with CRISPR-Cas9 to increase the yield of the recombinant protein. The epo gene was codon-optimized and its expression driven by the PmbfA promoter. Another version of epo contained introns from the fructose-1,6-bisphosphatase (fbp) gene. Two recombinant clones, uME12 (no introns) and uME23 (with introns), were selected based on the resistance to the antibiotic and because they showed a protein profile different from that of the parental strain, as shown by SDS-PAGE. Expression of epo was confirmed by RT-PCR in both colonies but the recombinant EPO protein (rHUEPO) was detected by Western blot only in uME23. The rHuEPO yield from uME23 was estimated at about 1.8 mg L−1 by ELISA, demonstrating that the presence of introns resulted in higher yield, possibly by conferring more stability to mRNA. On the other hand, as part of our strategy we decided to inactivate in the strain uME23 the following genes vps, prtT, algC and och1 which are involved in protein secretion, regulating of protease expression and protein glycosylation in A. niger, with CRISPR-Cas9, yielding the muPS20 transformant. muPS20 is a protease-free strain and its rHuEPO production level was increased 41.1-fold. Moreover, its molecular weight was ≈27 kDa showing that mutations in the above mentioned genes improved secretion, prevented proteolytic degradation and hyperglycosylation of heterologous protein.

TRNA introns: Presence, processing, and purpose.

The presence of introns in both protein-coding and noncoding RNA transcripts is a fascinating phenomenon. It seems counterintuitive that an organism would devote precious time and energy to removing a nucleic acid sequence that will not be present in the final product. Nevertheless, introns (including self-splicing ones) are clearly important components of the basic cellular process of gene expression. Transfer RNA (tRNA) introns have been detected in all three kingdoms of life, and their precise removal is crucial for tRNA function. Of particular interest to this review are the tRNA intronic circular RNAs (tricRNAs) that form during metazoan tRNA splicing. In animal cells, these ultrastable introns form a novel class of noncoding RNA. Here, we summarize established knowledge and describe new findings in the field of tRNA splicing. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA in Disease and Development > RNA in Disease RNA Processing > tRNA Processing.

Putative group I introns in the eukaryote nuclear internal transcribed spacers.

Group I introns are mobile genetic elements that interrupt genes encoding proteins and RNAs. In the rRNA operon, introns can insert in the small subunit (SSU) and large subunit (LSU) of a wide variety of protists and various prokaryotes, but they were never found in the ITS region. In this study, unusually long ITS regions of fungi and closely related unicellular organisms (Polychytrium aggregatum, Mitosporidium daphniae, Amoeboaphelidium occidentale and Nuclearia simplex) were analysed. While the insertion of repeats is responsible for long ITS in other eukaryotes, the increased size of the sequences analysed herein seems rather due to the presence of introns in ITS-1 or ITS-2. The identified insertions can be folded in secondary structures according to group I intron models, and they cluster within introns in conserved core-based phylogeny. In addition, for Mitosporidium, Amoeboaphelidium and Nuclearia, more conventional ITS-2 structures can be deduced once spacer introns are removed. Sequences of five shark species were also analysed for their structure and included in phylogeny because of unpublished work reporting introns in their ITS, obtaining congruent results. Overall, the data presented herein indicate that spacer regions may contain introns.

Understanding evolution in Poales: Insights from Eriocaulaceae plastome.

In this study, we report the plastome of Eriocaulon decemflorum (Eriocaulaceae) and make an effort to understand the genome evolution, structural rearrangements and gene content of the order Poales by comparing it with other available plastomes. The size of complete E. decemflorum plastome is 151,671 bp with an LSC (81,477bp), SSC (17,180bp) and a pair of IRs (26,507 bp). The plastome exhibits GC content of 35.8% and 134 protein-coding genes with 19 genes duplicated in the IR region. The Eriocaulaceae plastome is characterized by the presence of accD, ycf1 and ycf2 genes and presence of introns in clpP and rpoC1 genes which have been lost in the Graminid plastomes. Phylogenomic analysis based on 81 protein-coding genes placed Eriocaulaceae sister to Mayacaceae. The present study enhances our understanding of the evolution of Poales by analyzing the plastome data from the order.

CHO genome mining for synthetic promoter design

Synthetic promoters are an attractive alternative for use in mammalian hosts such as CHO cells as they can be designed de novo with user-defined functionalities. In this study, we describe and validate a method for bioprocess-directed design of synthetic promoters utilizing CHO genomic sequence information. We designed promoters with two objective features, (i) constitutive high-level recombinant gene transcription, and (ii) upregulated transcription under mild hypothermia or late-stage culture. CHO genes varying in transcriptional activity were selected based on a comparative analysis of RNA-Seq transcript levels in normal and biphasic cultures in combination with estimates of mRNA half-life from published genome scale datasets. Discrete transcription factor regulatory elements (TFREs) upstream of these genes were informatically identified and functionally screened in vitro to identify a subset of TFREs with the potential to support high activity recombinant gene transcription during biphasic cell culture processes. Two libraries of heterotypic synthetic promoters with varying TFRE combinations were then designed in silico that exhibited a maximal 2.5-fold increase in transcriptional strength over the CMV-IE promoter after transient transfection into host CHO-K1 cells. A subset of synthetic promoters was then used to create stable transfectant pools using CHO-K1 cells under glutamine synthetase selection. Whilst not achieving the maximal 2.5-fold increase in productivity over stable pools harboring the CMV promoter, all stably transfected cells utilizing synthetic promoters exhibited increased reporter production — up to 1.6-fold that of cells employing CMV, both in the presence or absence of intron A immediately downstream of the promoter. The increased productivity of stably transfected cells harboring synthetic promoters was maintained during fed-batch culture, with or without a transition to mild hypothermia at the onset of stationary phase. Our data exemplify that it is important to consider both host cell and intended bioprocess contexts as design criteria in the de novo construction of synthetic genetic parts for mammalian cell engineering.

Introns are mediators of cell response to starvation.

Introns are ubiquitous features of all eukaryotic cells. Introns need to be removed from nascent messenger RNA through the process of splicing to produce functional proteins. Here we show that the physical presence of introns in the genome promotes cell survival under starvation conditions. A systematic deletion set of all known introns in budding yeast genes indicates that, in most cases, cells with an intron deletion are impaired when nutrients are depleted. This effect of introns on growth is not linked to the expression of the host gene, and was reproduced even when translation of the host mRNA was blocked. Transcriptomic and genetic analyses indicate that introns promote resistance to starvation by enhancing the repression of ribosomal protein genes that are downstream of the nutrient-sensing TORC1 and PKA pathways. Our results reveal functions of introns that may help to explain their evolutionary preservation in genes, and uncover regulatory mechanisms of cell adaptations to starvation.